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Investigation of Receptor-Mediated Cyanocobalamin (Vitamin B12) Transport across the Inner Blood–Retinal Barrier Using Fluorescence-Labeled Cyanocobalamin
Molecular Pharmaceutics ( IF 4.5 ) Pub Date : 2018-07-02 00:00:00 , DOI: 10.1021/acs.molpharmaceut.8b00617
Yuri Kinoshita 1 , Kagayaki Nogami , Ryuta Jomura 1 , Shin-ichi Akanuma 1 , Hajime Abe , Masahiko Inouye , Yoshiyuki Kubo 1 , Ken-ichi Hosoya 1
Molecular Pharmaceutics ( IF 4.5 ) Pub Date : 2018-07-02 00:00:00 , DOI: 10.1021/acs.molpharmaceut.8b00617
Yuri Kinoshita 1 , Kagayaki Nogami , Ryuta Jomura 1 , Shin-ichi Akanuma 1 , Hajime Abe , Masahiko Inouye , Yoshiyuki Kubo 1 , Ken-ichi Hosoya 1
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The blood-to-retina supply of cyanocobalamin (vitamin B12) across the blood–retinal barrier (BRB) was investigated by synthesizing a fluorescence-labeled cyanocobalamin (Cy5-cyanocobalamin). In the in vivo analysis following internal jugular injection of Cy5-cyanocobalamin, confocal microscopy showed the distribution of Cy5-cyanocobalamin in the inner plexiform layer (IPL), the outer plexiform layer (OPL), and the retinal pigment epithelium (RPE). In the in vitro analysis with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, Cy5-cyanocobalamin uptake by TR-iBRB2 cells exhibited a time-dependent increase after preincubation with transcobalamin II (TCII) protein, during its residual uptake without preincubation with TCII protein. The Cy5-cyanocobalamin uptake by TR-iBRB2 cells was significantly reduced in the presence of unlabeled cyanocobalamin, chlorpromazine, and chloroquine and was also significantly reduced under Ca2+-free conditions. Confocal microscopy of the TR-iBRB2 cells showed fluorescence signals of Cy5-cyanocobalamin and GFP-TCII protein, and these signals merged with each other. The RT-PCR, Western blot, and immunohistochemistry clearly suggested the expression of TCII receptor (TCII-R) in the inner and outer BRB. These results suggested the involvement of receptor-mediated endocytosis in the blood-to-retina transport of cyanocobalamin at the inner BRB with implying its possible involvement at the outer BRB.
中文翻译：
用荧光标记的氰钴胺介导受体介导的氰钴胺（维生素B 12）跨内血-视网膜屏障的运输的研究
通过合成荧光标记的氰钴胺素（Cy5-cyanocobalamin），研究了跨血-视网膜屏障（BRB）的血液向视网膜中的氰钴胺素（维生素B 12）供应。在颈内注射Cy5-cyanocobalamin后的体内分析中，共聚焦显微镜显示Cy5-cyanocobalamin在内部丛状层（IPL），外部丛状层（OPL）和视网膜色素上皮（RPE）中的分布。在体外与TR-iBRB2细胞，分析在体外在内部BRB的模型细胞系中，TR-iBRB2细胞对Cy5-cyanocobalamin的摄取在与transcobalamin II（TCII）蛋白质预孵育后，在其残留摄取而未与TCII蛋白质预先孵育的过程中，表现出随时间的增加。在未标记的氰钴胺素，氯丙嗪和氯喹存在下，TR-iBRB2细胞对Cy5-cyanocobalamin的摄取显着降低，在Ca 2+下也显着降低无条件。TR-iBRB2细胞的共聚焦显微镜检查显示Cy5-cyanocobalamin和GFP-TCII蛋白的荧光信号，并且这些信号相互融合。RT-PCR，Western印迹和免疫组化清楚地表明了内，外BRB中TCII受体（TCII-R）的表达。这些结果表明，受体介导的内吞作用参与了内部BRB的氰钴胺素从血液到视网膜的运输，这暗示了其可能参与了外部BRB。
更新日期：2018-07-02
中文翻译：
用荧光标记的氰钴胺介导受体介导的氰钴胺（维生素B 12）跨内血-视网膜屏障的运输的研究
通过合成荧光标记的氰钴胺素（Cy5-cyanocobalamin），研究了跨血-视网膜屏障（BRB）的血液向视网膜中的氰钴胺素（维生素B 12）供应。在颈内注射Cy5-cyanocobalamin后的体内分析中，共聚焦显微镜显示Cy5-cyanocobalamin在内部丛状层（IPL），外部丛状层（OPL）和视网膜色素上皮（RPE）中的分布。在体外与TR-iBRB2细胞，分析在体外在内部BRB的模型细胞系中，TR-iBRB2细胞对Cy5-cyanocobalamin的摄取在与transcobalamin II（TCII）蛋白质预孵育后，在其残留摄取而未与TCII蛋白质预先孵育的过程中，表现出随时间的增加。在未标记的氰钴胺素，氯丙嗪和氯喹存在下，TR-iBRB2细胞对Cy5-cyanocobalamin的摄取显着降低，在Ca 2+下也显着降低无条件。TR-iBRB2细胞的共聚焦显微镜检查显示Cy5-cyanocobalamin和GFP-TCII蛋白的荧光信号，并且这些信号相互融合。RT-PCR，Western印迹和免疫组化清楚地表明了内，外BRB中TCII受体（TCII-R）的表达。这些结果表明，受体介导的内吞作用参与了内部BRB的氰钴胺素从血液到视网膜的运输，这暗示了其可能参与了外部BRB。
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